One of the principal mechanisms by which cellular regulation is effected is through the transduction of extracellular signals across the membrane that in turn modulate biochemical pathways within the cell. Protein phosphorylation represents one course by which intracellular signals are propagated from molecule to molecule resulting finally in a cellular response. These signal transduction cascades are highly regulated and often overlapping as evidenced by the existence of many protein kinases as well as phosphatases. It is currently believed that a number of disease states and/or disorders are a result of either aberrant expression or functional mutations in the molecular components of kinase cascades. Consequently, considerable attention has been devoted to the characterization of these proteins.
Nearly all cell surface receptors use one or more of the mitogen-activated protein kinase (MAP kinase) cascades during signal transduction. Three distinct subgroups of the MAP kinases have been identified and each of these consists of a specific module of downstream kinases. One subgroup of the MAP kinases is the Jun N-terminal kinase/Stress activated protein kinase (JNK/SAPK) cascade. This pathway was originally identified as an oncogene- and ultraviolet light-stimulated kinase pathway but is now known to be activated by growth factors, cytokines and T-cell costimulation.
Jun N-terminal Kinase Kinase-2 (also known as JNKK2, PRKMK7, MAPKK7 and MKK7) is a dual-specificity MAP kinase kinase that acts to mediate signaling pathways initiated by cellular stressors. The enzyme is ubiquitously expressed with the highest expression found in heart and skeletal muscle (Foltz et al., J. Biol. Chem., 1998, 273, 9344-9351; Yang et al., Gene, 1998, 212, 95-102).
Six murine (Tournier et al., Mol. Cell. Biol., 1999, 19, 1569-1581) and four human isoforms of JNKK2 have been isolated (Foltz et al., J. Biol. Chem., 1998, 273, 9344-9351; Lu et al., J. Biol. Chem., 1997, 272, 24751-24754; Wu et al., Mol. Cell. Biol., 1997, 17, 7407-7416; Yang et al., Gene, 1998, 212, 95-102) and polynucleotides encoding the human isoforms are disclosed in PCT publication WO 99/02457 (Davis et al.). Also disclosed are vectors and host cells that express JNKK2 and methods of measuring and modulating JNKK2.
JNKK2 has been shown to be regulated and activated to varying degrees by ultraviolet exposure (Butterfield et al., Biochem. J., 1999, 338, 681-686), the .beta..gamma. subunit of G-proteins (Yamauchi et al., J. Biol. Chem., 1999, 274, 1957-1965) and mixed lineage kinase-2 (Cuenda and Dorow, Biochem. J., 1998, 333, 11-15; Hirai et al., J. Biol. Chem., 1998, 273, 7406-7412) suggesting that specific stresses affect independent signaling pathways through the various isoforms. JNKK2 has been shown to act in select cascades, being significantly activated by Rac and Cdc42Hs two upstream kinases that are members of the Rho small GTP-binding protein family as well as tumor necrosis factor .alpha. (Lu et al., J. Biol. Chem., 1997, 272, 24751-24754) and Fas, a membrane protein that transduces apoptotic signals to the cytoplasm (Toyoshima et al., J. Cell. Biol., 1997, 139, 1005-1015). Recently JNKK2 was shown to be activated during T-cell activation with the inhibition of this MAP kinase pathway resulting in blocked interleukin-2 transcription. These results suggest a role for JNKK2 in the development and proliferation of T-lymphocytes (Matsuda et al., J. Biol. Chem., 1998, 273, 12378-12382).
To date, strategies aimed at inhibiting JNKK2 function have involved the use of antibodies, dominant-negative forms of JNKK2 and SAPK pathway inhibitors. Disclosed in the PCT application WO 98/54203 (Mercola) are inhibitors of the SAPK pathway comprising antisense oligonucleotides to an upstream kinase, MEKK1, as well as antisense oligonucleotides targeting the kinase isoforms that lie downstream of JNKK2, SAPK1-3. Also disclosed are methods to inhibit the SAPK pathway using ribozymes, small molecule inhibitors and dominant-negative mutants.
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of JNKK2 and consequently, there remains a long felt need for additional agents capable of effectively inhibiting JNKK2 function.
Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of JNKK2 expression.